Fatty acid photodecarboxylase (FAP) is one of the few photoenzymes in nature. The ability of FAP to convert fatty acids into alka(e)nes without the need for reducing equivalents put this enzyme into spotlight for biocatalytic applications. Although it has been discovered only a few years ago, many studies already emerged demonstrating its potential in areas from biofuel production and enzymatic kinetic resolution to being a critical component of multi-enzyme cascades. While there have been few protein engineering studies for modulating activity of FAP towards very short chain fatty acids, no study has yet addressed substrate selectivity within the medium to long chain fatty acid range, where FAP shows great promise for the synthesis of drop-in biofuels from ubiquitous fatty acids with chain lengths from C12 to C18. Here, after determining optimum expression and assay conditions for FAP, we screened 22 rationally designed mutant enzymes towards four naturally abundant fatty acid substrates; C12 : 0, C16 : 0, C18 : 0 and C18 : 1. Depending on the type of the exchanged amino acid, we observed selectivity shifts towards shorter or longer chains, compared to wild type enzyme. Notably, we obtained two groups of mutants; one group with high selectivity towards only C18 : 0, and another group that is selective towards C12 : 0 substrate. Moreover, we measured light and thermal stability of the wild type enzyme as well as the light stability of a mutant engineered for selectivity.

The use of neat reaction media, that is the avoidance of additional solvents, is the simplest and the most efficient approach to follow in biocatalysis. Here, we show that unspecific peroxygenase from Agrocybe aegerita (AaeUPO) can hydroxylate the neat model substrate cyclohexane. H₂O₂ was photocatalytically generated in situ by nitrogen-doped carbon nanodots (N−CNDs) and UV LED illumination. AaeUPO entrapment in alginate beads increased enzyme stability and facilitated the reaction in neat cyclohexane. N−CNDs absorption in beads containing AaeUPO created a 2-in-1 heterogeneous photobiocatalyst that was active for up to seven days under reaction conditions and produced cyclohexanol, 2.5 mM. To increase productivity, the bead size and the photocatalyst-to-enzyme ratio have been identified as promising targets for optimisation.

A direct synthesis of lactams (5-, 6-, and 7-membered) starting from amino-alcohols in a bienzymatic cascade is reported. Horse liver alcohol dehydrogenase together with the NADH oxidase from Streptococcus mutans were applied for the oxidative lactamization of various amino alcohols. Crucial parameters for the efficiency of this cascade reaction were elucidated. This report represents a direct approach for biocatalytic oxidative lactamization reaction.

A nicotinamide adenine dinucleotide (NADH)-dependent redox-neutral convergent cascade composed of a recently discovered type II flavin-containing monooxygenase (FMO−E) and horse liver alcohol dehydrogenase (HLADH) has been established. Two model reaction cascades were analyzed for the synthesis of γ-butyrolactone and chiral bicyclic lactones. In the former cascade, all substrates were converted into one single product γ-butyrolactone with high atom efficiency. More than 130 mM γ-butyrolactone were obtained when applying 100 mM cyclobutanone and 50 mM 1,4-butanediol in this cascade. In the second cascade where bicyclo[4.2.0]octan-7-one and cis-1,2-cyclohexanedimethanol were coupled, the ketone substrate was converted to the corresponding normal lactone with an ee value of 89–74% (3aS, 7aS) by FMO−E alone and the abnormal lactone with an ee value of >99% (3aR, 7aS) was formed by both HLADH and FMO−E. (Figure presented.).

A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215–1221.